Lighting up the lightning speed of vesicle formation

When neurons throughout the brain and body send messages, they release chemical signals. These chemicals, neurotransmitters, pass into the spaces between neurons, or synapses, binding to receptors to send a signal along. When they are not in use, neurotransmitters are stored within the cell in tiny bubbles called vesicles. During signaling, these vesicles head to the membrane of the neuron, where they dump neurotransmitter into the synapse. And after delivering their cargo, most vesicles disappear. But more vesicles keep forming, filling with neurotransmitters so neurons can keep sending signals. What goes up must come down. When vesicles go out, they must come back.

But how fast do the vesicles re-appear? Much faster, it turns out, than we first thought.

Neurotransmission happens fast. An electrical signal comes down a neuron in your brain and triggers vesicles to move to the cell membrane. When the vesicles merge into the membrane and release their chemical cargo, the neurotransmitters float across the open synapse to the next neuron. This happens every time the neuron “fires.” This needs to happen very quickly, as neurons often fire at 100 hertz, or 100 times per second.

Some vesicles perform a “kiss-and-run,” opening up a temporary pore in the membrane, releasing a little bit of neurotransmitter and darting away again. Other vesicles need to merge with the synapse entirely. With the assistance of docking proteins, these vesicles fuse with the membrane of the neuron to release the neurotransmitters, a process called exocytosis.

In essence, these vesicles disappear. But of course, neurons need new vesicles to stay loaded to send signals. New vesicles form through a process called, you guessed it, endocytosis. A protein called clathrin helps create a bulge in the membrane that pinches off to make a new vesicle. But endocytosis using clathrin takes several seconds (an eternity in cellular terms). And this is at odds with the huge speed of neurotransmitter release. If a neuron is firing 100 times per second, vesicles are merging and kissing and running all over the place. All the merging is going to deplete the readily available vesicle pool and rapidly expand the area of the membrane. There is going to be a need for speedy endocytosis to make more vesicles and to keep the membrane area down.

Shigeki Watanabe and colleagues from the United States and Germany wanted to find out how fast endocytosis could occur, and if it happened at speed, what helped it along. It turns out that clathrin isn’t the only game in town. Endocytosis can happen a lot faster than we might have supposed.

They found this out using optogenetics, a technique that puts proteins that respond to light into cells — in this case, mouse brain cells in a dish. When the proteins, called channelrhodopsins, are in the neurons, a brief burst of light makes the channelrhodopsin respond. The channel in the protein opens, producing a change in potential that makes the neurons fire. They coupled the optogenetics with another technique that allowed them to stop the neurons in time, milliseconds after the neurons had fired. By using this “flash and freeze” technique, the authors could get snapshots of the synapse, from the moment of firing to a second or so afterward. This allowed them to count the vesicles that were merging as well as the ones that were forming, to see where and how fast endocytosis and exocytosis were taking place.

In a paper published December 5 in Nature, the authors show that, in fact, membranes are capable of making vesicles much, much faster than previously known. Vesicle endocytosis can occur with 100 milliseconds of a single stimulation. Vesicle release occurs within about 30 milliseconds. And it occurs very close to the active zone where vesicle release is taking place. There is so much endocytosis going on that it almost keeps up with the vesicular release. The authors call this “ultrafast endocytosis.”

But what is controlling this endocytosis? Clathrin, as I mentioned above, is slow and takes several seconds to invaginate (that’s the technical term) the membrane to form a vesicle. Instead, the authors found that two other proteins were involved, actin and dynamin. Actin formed the inward bulge of the membrane, and dynamin helped pinch the new vesicles off.

So it’s not all slow endocytosis, and vesicle formation is not all clathrin. The new study adds another layer of complexity to our understanding of the synapse and how neuronal communication works, and shows that what goes out must come back in, and very quickly indeed.